Purification of pertussis antigens

Abstract

A process is provided for the production of lymphocytosis promoting factor (LPF), filamentous haemagglutinin (FHA) and at least one fimbrial agglutinogen from a liquid culture of Bordetella pertussis, which comprises the steps of (a) separating the culture into cellular and supernatant fractions, (b) concentrating the supernatant fraction, (c) fractionating the concentrated supernatant fraction to isolate LPF and FHA containing fractions, and (d) isolating at least one fimbrial agglutinogen from the cellular fraction. A vaccine composition may be produced by mixing so-produced LPF, FHA and fimbrial agglutinogens produced.

We claim: 
     
       1. A process for the co-production of lymphocytosis promoting factor (LPF), filamentous haemagglutinin (FHA) and at least one fimbrial agglutinogen from a liquid culture of Bordetella pertussis, which comprises the steps of (a) separating the culture into cellular and supernatant fractions,   (b) subsequent to step (a) concentrating the supernatant fraction to less than 50% of its original volume to form a concentrated aqueous supernatant fraction of reduced water content,   (c) subsequent to step (b), fractionating the concentrated supernatant fraction to isolate LPF and FHA containing fractions, and   (d) isolating at least one fimbrial agglutinogen from the cellular fraction.   
     
     
       2. A process according to claim 1 wherein the fimbrial agglutinogens isolated in step (d) include at least one of agglutinogens 2, 3, 4, 5 and 6. 
     
     
       3. A process according to claim 1 wherein the fimbrial agglutinogens isolated in step (d) comprise at least agglutinogens 2 and 3 (Ag 2+3 ). 
     
     
       4. A process according to claim 3 wherein the isolated agglutinogens additionally include one or more of agglutinogens 4, 5 and 6. 
     
     
       5. A process according to claim 1 wherein separation step (a) is carried out at a pH greater than 7.0. 
     
     
       6. A process according to claim 5 wherein separation step (a) is carried out at a pH in the range from 7.5 to 9.0. 
     
     
       7. A process according to claim 1 wherein in step (b) the supernatant is concentrated to less than 50% and preferably less than 25% of its original volume. 
     
     
       8. A process according to claim 1 wherein in step (c) the LPF containing fraction is purified by adsorption on fetuin sepharose followed by elution using a magnesium chloride buffer. 
     
     
       9. A process for the production of a vaccine composition comprising lymphocytosis promoting factor (LPF), filamentous haemagglutinin (FHA) and at least one fimbrial agglutinogen which comprises mixing (i) LPF, (ii) FHA and (iii) at least one fimbrial agglutinogens produced according to claim 1, the LPF, FHA and the at least one fimbrial agglutinogens being detoxified prior to or subsequent to mixing.